The FixL protein is a biological sensor of O2. Dissociation of O2 from a heme-binding domain is required for FixL to autophosphorylate at a conserved histidine with a gamma-phosphoryl group from ATP. Transfer of the phosphoryl group from FixL to the transcription factor FixJ enables FixJ to activate a cascade of gene expression. The kinase domain of FixL belongs to a large class of sensory histidine kinases that occur in plants, fungi, Archaea, and every bacterium that has been examined. The structure of such a kinase domain has recently become available. Crystal structures were also recently solved for the heme-binding domain of FixL with and without an inhibitory ligand. These FixL structures have led to a new model for heme-driven conformational changes. Although the alpha/beta fold of the FixL, heme-binding domain represents a novel hemoglobin fold, this fold closely matches the structures of the HERG voltage sensory in humans and the PYP light sensory in halophilic bacterium. As such, the heme-binding domain of FixL provides a prototype for a large family of sensory proteins called the PAS proteins. Based on sequence alignments, over 200 PAS proteins have been identified so far in Archaea, Bacteria, and Eukarya, where they transduce key regulatory signals. The direct coupling in FixL of a histidine kinase to an O2-detecting PAS domain provides an ideal system for study of signal transduction. The proposed work will combined mutagenesis with biochemical, biophysical, and structural approaches to: test the regulatory mechanism indicated by the FixL "on" and "off" structures, examine the role of conserved PAS residues, determine factors that govern affinity for ligands, and identify key contacts between the heme-binding and kinase domains of FixL. This research is expected to extend knowledge of signal transduction mechanisms and sensing of physiological heme ligands. Some possible applications are the development of novel antibiotics directed at specific sensors in bacteria and anti-cancer drugs targeting O2 sensors that are implicated in angiogenesis.